African trypanosomes are parasitic protozoa and causative pathogens of sleeping sickness in humans and nagana in cattle. They are deeply branched primitive eukaryotes with many simple and unique biological features to make interesting models for basic studies and provide opportunities for chemotherapeutic control of the diseases they cause. The 26S proteasome in Trypanosoma brucei exists in a single species and performs many vital functions including cell cycle regulation. Its substrate specificity differs from that of the human proteasome, which allows specific inhibitor design. Insect form T. brucei has also a very simple cyclin-mediated cell cycle control involving only one essential G1 cyclin and one mitotic cyclin resulting in a leaky checkpoint regulation. There are four specific aims in our proposal; (1) to analyze the activity profiles of individual catalytic subunits in the T. brucei proteasome and compare them with those of the human proteasome. A P1-glutamine tetrapeptide has been identified as a preferred substrate by the T. brucei proteasome. Further selective substrate and inhibitor design will be pursued with the increasing understanding of proteasome activity profiles; (2) to crystallize the 19S regulatory complex of T. brucei proteasome for structural analysis. The complex has an extraordinary stability and could become the first to have its structure resolved; (3) to analyze the G2/M checkpoint regulation in the bloodstream and insect forms of T. brucei. A regulatory complex most likely consisting of the mitotic cyclin, cyclin-dependent protein kinase, anaphase promoting complex (E3), an E2 and the proteasome will be identified and analyzed by the tandem affinity purification system and yeast two-hybrid screen and the functions of individual subunits characterized by RNA interference analysis; (4) to analyze and compare the two different mechanisms of G1/S checkpoint control in the bloodstream and insect forms of T. brucei and identify the two distinctive regulatory complexes by similar experimental approaches as stated above. The two forms of T. brucei share a similar G2/M control but distinctive G1/S regulations. A potential shift from one machinery of G1/S control to another during the differentiation from bloodstream into the insect form will be closely monitored and the triggering factor for this shift will be pursued.